1. Field of the Invention
The present invention relates to a method for obtaining information about an analyte using a time of flight mass spectrometer, and more particularly, to a method for imaging detection of components, such as proteins or other organic substances, of the analyte on a type by type basis.
2. Description of the Related Art
Recently, with advances in genome analysis, increasing importance is given to techniques for analyzing proteins which are gene products existing in a living body, and more particularly, techniques for visualization of protein chips or proteins which have a distribution state such as found in living tissue.
Conventionally, importance of expression and functional analysis of proteins have been pointed out and analysis techniques have been developed. Basically, such techniques are based on a combination of: (1) separation and refining by means of two-dimensional electrophoresis or high-performance liquid chromatography (HPLC) and (2) a detection system including radiation analysis, optical analysis and mass spectrometry.
Development of a protein analysis technique is roughly divided into database construction by means of proteome analysis (comprehensive analysis of intracellular proteins) which, in a sense, is the basics of protein analysis and development of diagnostic devices or drug discovery devices (drug-candidate screening) devices based on the resulting database. However, conventional methods often leave problems in terms of analysis time, throughput, sensitivity, resolution and flexibility. In any application, there is demand for devices different from the conventional methods with such problems and suitable for downsizing, speedup and automation. To meet this demand, development of a so-called protein chip in which protein is packed densely has been drawing attention. The protein chip is formed by fixing protein which will serve as a probe to a substrate surface and forming an organic film around the fixed protein to prevent nonspecific absorption. Then, the protein chip is used for diagnosis or screening by pouring a solution containing a target drug candidate onto the protein chip and assessing an amount of absorption through an antigen-antibody reaction.
However, there is no way to evaluate precisely whether the protein chip has been formed properly because it is difficult to obtain two-dimensional distribution of the protein in a minute area at the current level of technology.
In mass spectrometry (MS) of proteins, time of flight secondary ion mass spectrometry (hereinafter abbreviated to TOF-SIMS) has come to be used recently as a means of high-sensitivity mass analysis or as a means of surface analysis.
The TOF-SIMS is an analysis method which is used to check what atoms or molecules are present on an outermost surface of a solid sample. The method has the following features: (1) capable of detecting components in trace amounts on the order of 109 atoms/cm2; (quantity corresponding to 1/105 of the outermost surface mono atomic layer); (2) applicable to both organic and inorganic substances and capable of measuring all elements and compounds on the surface; and (3) capable of secondary-ion imaging from substances existing on the sample surface.
Principles of the method will be described briefly below.
When a high-speed pulsed ion beam (primary ions) is directed onto a surface of a solid sample in a high vacuum, components on the surface are released into the vacuum by sputtering. Positively or negatively charged ions (secondary ions) thus generated are focused in one direction by means of an electric field and detected at a location some distance away. When primary ions are directed at a solid surface in a pulsed manner, secondary ions with various masses are generated depending on the composition of the sample surface. In so doing, lighter ions fly faster and heavier ions fly more slowly. Thus, the masses of the generated secondary ions can be analyzed by measuring the time (time of flight) required for the secondary ions to be detected after being generated. When the primary ions are directed onto a solid sample surface, only the secondary ions generated in the outermost layer of the solid sample surface are released into the vacuum, which provides information about the outermost surface (approximately a few Angstroms deep) of the sample. Since TOF-SIMS uses an extremely small dose of primary-ion irradiation, organic compounds are ionized with their chemical structures maintained, allowing the structures of the organic compounds to be learned from a mass spectrum. However, when subjected to TOF-SIMS under normal conditions, artificial polymers such as polyethylene or polyester or biopolymers such as protein are broken down into small fragment ions, making it difficult to know the original structures. On the other hand, when the solid sample is an insulator, the solid sample can be analyzed because positive charge accumulated on the solid surface can be neutralized by pulses of an electron beam directed at interstices among the primary ions emitted in a pulsed manner. In addition, TOF-SIMS allows an ion image (mapping) on the sample surface to be measured by scanning the sample surface with a primary-ion beam.
Examples of protein analysis using TOF-SIMS include a method which detects parent protein molecules of a high molecular weight by mixing the protein with a matrix substance using a pre-processing process similar to a MALDI process (Kuang Jen Wu et al., Anal. Chem., 68, 873, (1996)). Also, there is a method in which part of a particular protein is labeled with an isotope such as 15N and the protein is detected by imaging using secondary ions such as C15N− (A. M. Belu et al., Anal. Chem., 73, 143, (2001)). Furthermore, there are a method which estimates types of protein from types and relative strength of fragment ions (secondary ions) corresponding to amino acid residues (D. S. Mantus et al., Anal. Chem., 65, 1431, (1993)) and a method which determines detection limits of TOF-SIMS with respect to proteins absorbed by various substrates (M. S. Wagner et al., J. Biomater. Sci. Polymer Edn., 13, 407, (2002)).
Other mass spectrometric methods for protein include a method which uses field emission. The method causes the protein to form a coordinate or covalent bond on a metal electrode via an open group which can be split according to applied energy and then leads the protein to a mass spectrometer by the application of an intense electric field.
As described above, various methods have been proposed for analyzing a distribution state of a plurality of proteins contained in an analyte using mass spectrometry. However, since conventional mass spectrometry analyzes proteins or the like eluted from living tissue or protein chips by means of an appropriate solvent rather than analyzing the subject component itself, there is a limit to obtaining original distribution information about a sample. Also, the conventional mass spectrometry, with which it is difficult to know the distribution state of the proteins serving as a probe, cannot directly assess nonspecific absorption into chip surfaces.
The MALDI process and a SELDI process which is a modification of the MALDI process are the most flexible ionization method known today and have the excellent feature of being able to ionize degradable proteins of high molecular weight as they are and detect parent ions or equivalent ions. Currently, the MALDI and SELDI processes are one of standard ionization methods for mass spectrometry of proteins. On the other hand, when the methods are used for mass spectrometry of protein chips, the existence of a matrix substance makes it difficult to obtain a two-dimensional distribution image (imaging based on mass information) of protein with a high spatial resolution. That is, although a laser beam itself used as an excitation source can be focused into a diameter of approximately 1 to 2 μm, vaporization and ionization of the matrix substance existing around the protein to be analyzed are unavoidable even under laser irradiation with such a small spot. Under these circumstances, when a two-dimensional distribution image of protein is measured by any of the above methods, spatial resolution is generally somewhere around 100 μm. For scanning with the focused laser, it is necessary to move lenses and mirrors in a complicated manner. That is, when measuring a two-dimensional distribution image of protein by any of the above methods, it is generally difficult to scan a laser beam and only available method is to move a stage with a test sample mounted. To obtain a two-dimensional distribution image of protein by increasing spatial resolution, a method which involves moving a sample stage is not advisable.
Furthermore, in addition to the problem in obtaining a two-dimensional distribution image of protein by increasing spatial resolution, there are restrictions on the form of samples to be analyzed, such as a need to fix an object on a metal electrode.
Compared to the above methods, the TOF-SIMS technique, which uses primary ions, can easily focus and scan the primary ions and is suitable for obtaining a two-dimensional ion image (two-dimensional distribution image) of a high spatial resolution. The TOF-SIMS technique provides a spatial resolution of somewhere around 1 μm. However, when the analyte is protein or an inorganic compound, TOF-SIMS measurements under normal conditions mostly produce small fragment ions as secondary ions, making it generally difficult to know the original structure, as described above. Thus, in dealing with a sample such as a protein chip in which a plurality of proteins are arranged on a substrate, some measures must be devised to obtain a two-dimensional ion image (two-dimensional distribution image) of a high spatial resolution which will allow the types of the proteins to be identified. The method proposed by Kuang Jen Wu et al. can suppress degradation due to irradiation with primary ions even in the case of protein of high molecular weight and thereby detect the parent molecules while maintaining original mass. However, the method uses a mixture of protein and a matrix substance and cannot obtain original two-dimensional distribution information in the case of a sample such as the protein chip. Since the method proposed by A. M. Belu et al. isotope-labels part of a particular protein, the method can make full use of the high spatial resolution of TOF-SIMS. On the other hand, the method must isotope-label the particular protein every time. Also, with the method proposed by D. S. Mantus et al., i.e., the method which estimates types of protein from types and relative strength of fragment ions (secondary ions) corresponding to an amino acid residues, it may be difficult to determined the types of protein if there coexist proteins with similar amino acid structures.
If the TOF-SIMS technique is applied, with peptide chains of the protein molecules being held together, for example, to protein molecules in living tissue, production efficiency of secondary ion species is greatly reduced. For measurements by means of the TOF-SIMS technique, which involves primary-ion irradiation in a high vacuum, a sample to be measured is dried in advance. During the drying, if protein molecules and other biological material in the living tissue interact with each other and aggregate due to intermolecular bonding, the production efficiency of the secondary ion species is reduced further.
To analyze quantity of particular protein molecules present in living tissue with high detection sensitivity at a high level of quantification and perform two-dimensional imaging concerning quantity distribution of the particular protein molecules in a cut surface of the living tissue, it is important to solve the problem of holding of protein molecules. In other words, it is important to allow ions to fly efficiently by slowly releasing the protein molecules held together in the living tissue by ion sputtering through primary-ion irradiation. Such a flying state allows secondary ion species of effective parent molecules to be produced reliably.